In mammalian embryos, hemangioblasts are believed to be the precursors of angioblasts and totipotent or pluripotent hematopoietic progenitor (i.e. stem) cells. Angioblasts and other embryonic totipotent and/or pluripotent progenitor cells are believed to be the precursors of postnatal endothelial cells, muscle cells, and neural cells. Despite considerable progress, uncertainties regarding these systems remain.
In the hematopoietic system, pluripotent stem cells are believed to be able to repopulate all of the blood cell lineages in an ablated mammal. Various surface markers may be used to obtain purified populations of such stem cells.
For example, a purified population of CD34+ hematopoietic stem cells was described by Civin in U.S. Pat. Nos. 5,035,994 and 5,130,144. A more highly purified population of hematopoietic stem cells that are CD34+, Class II HLA+, and Thy-1+ was described by Tsukamoto et al. in U.S. Pat. No. 5,061,620.
The Tsukamoto patent further explains that stem cells lack certain markers that are characteristic of more mature, lineage-committed (Lin+) cells. Such markers include CD3, CD8, CD10, CD19, CD20, and CD33. Cells that lack these markers are said to be lineage negative (Lin−).
The development of the initial blood vessel system in embryos is generally believed to occur from the adhesion to each other and modeling of primitive endothelial precursor cells, such as angioblasts. This process is known as vasculogenesis.
Postnatal development of new blood vessels is generally believed to occur from the proliferation, migration, and remodeling of the mature endothelial cells of pre-existing blood vessels. This process is known as angiogenesis.
It has been suggested that angioblasts and hematopoietic stem cells share certain surface markers, such as CD34 and the FLK-1 receptor. The FLK-1 receptor is also known as vascular endothelial growth factor receptor-2 (VEGFR-2) and, in the case of the human receptor, KDR. These suggestions have led to speculation that CD34+ mononuclear blood cells isolated from human peripheral blood may contribute to neoangiogenesis. See, for example, Pepper, Arteriosclerosis, Thrombosis, and Vascular Biology 17, 605-619 (April, 1997); Asahara et al., Science 275, 964-967 (Feb. 14, 1997).
There have been no reports that establish with confidence the existence of a population of endothelial, muscle, or neural progenitor cells comparable to hematopoietic progenitor cells, or, a fortiori, a method of isolating and purifying such cells.
Little is known with confidence, moreover, about the surface markers that differentiate endothelial progenitor cells from mature cells. For example, although CD34 appears to be a surface marker on endothelial progenitor cells, some mature endothelial cells also are CD34+.
The lack of information regarding surface markers on endothelial, muscle, or neural progenitor cells has made it difficult to isolate purified populations of these cells that can be used for therapeutic purposes. Such populations of progenitor cells are believed to be recruited at sites of cell growth and organ formation, at least in embryos. Less is known about the development of new cells and organs in adults.
The object of the present invention is to provide purified populations of endothelial, muscle, and neural stem cells. Another object of the present invention is to provide methods for isolating such stem cells. Another object of the present invention is to provide methods whereby populations of endothelial, muscle, and neural stem cells can be used in the treatment of conditions that require neovascularization, neomyogenesis, and neoneurogenesis, as well as in gene therapy.